The analytical determination of phosphorus containing chemically complex molecules is fraught with serious difficulties due mainly to their physicochemical properties. These molecules are highly charged and due to the presence of complex functional groups, such as phosphates or phosphonates, show a different ionization degree depending on pH. This issue becomes especially relevant for molecules with more than one phosphorus containing group.
Likewise, the absorption bands lack within the UV-Vis spectrum region in the majority of these chemically complex molecules hinder its quantification by direct spectrophotometric methods.
Furthermore, the determination of chemically complex molecules within a biological matrix involves additional difficulties due to the matrix effect, so new bioanalytical methods for routine use must be developed and validated.
The low concentration of the analyte compared to other components in the matrix can suppress the analyte response. These effects can cause differences in response between sample in matrix and standards, leading to difficulties in quantitative analysis and compound identification (Biol Pharm Bull 25, 547-557; 2002).
For example, bisphosphonates present two phosphonic groups per molecule which lend a strongly ionic character and increased polarity. Additionally, the majority of the members of this family lacks of chromophores excluding convenient direct UV detection (J Pharm Biomed Anal 48, 483-496; 2008). Other bioanalytical methods have been developed for the determination of bisphosphonates, adding a derivatization step for its determination or fragmentation not being a direct method, understood direct method as the determination of the molecule per se or an adduct of this molecule, what provides a more sensible, specific, accurate, and robust method but specially more applicable, quick and reliable methodologies for routine analysis (J Chromatogr B 877, 3159-3168; 2009, Int J Mass Spectrom 295, 85-93; 2010, J Mass Spectrom 37, 197-208; 2002).
Other example of phosphorus containing chemically complex molecule are pyrophosphates. Pyrophosphates differ from the bisphosphonates in the carbon atom that binds the two phosphorus atoms (Carbon atom of the bisphosphonates (P—C—P) is replaced by an oxygen (P—O—P)).
Nucleotides are also an example of chemically complex molecules, highly charged and with polar nature (due to the presence of one or more phosphate groups), thus being this invention especially relevant for those with more than one phosphate group.
The selective determination of inositol polyphosphates (from 2 to 6 phosphate groups), together with other related impurities and especially in biological matrices is one of the major analytical breakthroughs faced in this invention. In the extreme case, inositol hexaphosphate, also known as InsP6 or IP6, presents 12 dissociable protons, having pKa values that range from negative values to more than 10 (Carbohydr Res 46, 159-171; 1976).
Numerous analytical methods for InsP6 quantification have been described in bibliography. Nevertheless, most of them have been developed for determination in simple matrices, (e.g. those that come from methods for food extracts or pharmaceutical preparations), where concentrations of InsP6 are higher than expected in biological matrices.
In the case of complex matrices (generally, those with biological origin) previous cited methods have not sensitivity and specificity enough for InsP6 determination when concentrations to be quantified are low, as the case of plasma, urine, other biological fluids, tissues or cells.
A high sensible and selective method is needed for the quantification of inositol polyphosphates in this kind of matrices, and together with their special physicochemical properties, make most of the current methods useless.
Moreover, although some of the current methods could detect inositol polyphosphates, they are not reproducible and solid enough for their use in clinical studies, when routine sample analysis must be performed.
The WO/2009/109647 discloses the determination of the amount of inositol phosphate (IP1 or InsP1, only 1 phosphate group) in the sample (urine, plasma) by using LC-ESI/MS/MS assay. The US20100136600 discloses the determination of myo-inositol in the sample (urine, plasma) by using LC-MS/HPLC-MS techniques. However, neither of them disclose the IP6 determination method and also the determination technique disclosed in these patents may not be effective/suitable for IP2-IP6 determination because IP2-IP6 are highly charged molecules as compared to the inositol (no phosphate groups, no charge) or IP1 (only one phosphate group, limited charge).
Indirect methods for the determination of InsP6 and InsP3 in plasma were previously developed by using gas chromatography-mass detection analysis of HPLC chromatographic fractions which involves hydrolysis of IP6 and further, determination of inositol or phosphate, leading to a highly time consuming methodology (3 days per sample) and poorly accurate results (Life Sci 71, 1535-1546; 2002).
Direct HPLC-MS for its quantification in plants extracts and in vitro culture cells have already been described (Mass Spectrom 23, 705-712; 2009). However, the HPLC conditions described make the method useless for all the other biological matrices.
HPLC/MS with thermospray ionization allows the determination of inositol phosphates, but the lack of sensitivity is stated in the paper as a limitation for biological applications (Biomed Environ Mass Spectrom 19, 597-600; 1990).
Other indirect methods for the determination of IP6 in human urine are based on the total phosphorus measurement of purified extracts of phytic acid are described. In this case, a specific pretreatment of the sample is required to avoid interference from other phosphorus containing compounds accompanying phytic acid in urine such as phosphate or pyrophosphate (Anal Chem 75, 6374-6378; 2003, Anal Chim Acta 510, 41-43; 2004). One of the drawbacks of these methods is that they are limited to, due to sensitivity and selectivity issues, urine samples, not being applicable to tissues or blood samples and to the quantification of related impurities. In addition, the indirect determination of IP6 through total phosphorus is an extrapolation which leads to systematic high values since all the quantified phosphorus is identified as IP6.
Another indirect method is described for the determination of phytate in human urine. The method is based on hydrolysis of the phytate and determination of myo-inositol, one of the hydrolysis products (Chromatographia 60, 265-268; 2004). This is a time-consuming methodology (2 days per sample) due to the need of acidic hydrolysis. Furthermore, the limited sensitivity and the lack of specificity of the hydrolysis make the method useless for other biological matrices and for the co-quantification of related impurities.
Other documents considered as prior art for IP6 determination in biological samples (mainly in urine) are cited below:
March et al described in 2001 a unique methodology to quantify inositol phosphates in blood, but also applicable to urine and tissues (J Chromatogr B 757, 247-255; 2001). It's a time-consuming indirect methodology (3 days per sample) based on the enzymatic hydrolysis of IP6 and determination of the inositol molecule (hydrolysate) through gas chromatography and mass spectrometry. It's a sensitive method, although indirect, based on different principles from the present invention, which in any case fails in the specificity, since all inositol phosphates are hydrolysed inespecifically and the whole hydrolysate is quantified as IP6.
Fluorescence has been widely used for the quantification of IP6, although the poor sensitivity just allows the application in foods and in urine as biological samples (Anal Chim Acta 605, 185-191; 2007).
They are totally different methods from the invention here described, which in any case fail to determine related impurities and are not applicable to other biological matrices different from urine.